Movement of ions and small solutes across endothelium and epithelium of perfused rabbit lungs

In situ and isolated fluid-filled rabbit lungs were used to study the transport of indicators between the air space and vascular compartments. These indicators were placed in either the perfusate or air spaces and samples were collected from the perfusate at intervals during a 1-h perfusion period. At the end of the hour, fluid was pumped out of the air space compartment into serial tubes and indicator concentrations were determined in both the air space and perfusion fluids. One hour after introducing the indicators into the air space, the relative decreases in solute concentration were (arranged from the greatest to the least decline): [14C]urea greater than 36Cl- = 125I- greater than 22Na+ greater than [3H]mannitol. The relative rates at which the indicators appeared in the perfusate were similar. When the indicators were placed in the perfusate, a similar relationship was observed in the increase in air space concentrations, but the loss of 22Na+ from the perfusate was similar to those of 36Cl- and 125I-. Losses of all indicators from the perfusate were two or more times those from the air spaces, and although the loss of [3H]mannitol from the perfusate was similar to that of 22Na+ for about 30 min, subsequent loss was much slower. Very little 125I-albumin traversed the tissue barrier, and the small changes in the concentrations of 125I-albumin in the air spaces suggested that little fluid movement had occurred. These studies suggest that the epithelium is less permeable to solutes than the endothelium and permits passage of anions at a faster rate than 22Na+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular release of nonheme iron in perfused rabbit lungs

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O(2)) or hypoxic (95% N(2)) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released approximately 0.25 microg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by approximately 50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and approximately 60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.     

Leaky intra-acinar arteries in rat lungs perfused with hydrogen peroxide

We have investigated the effects of H2O2 (150 or 300 microM) on the ultrastructure and permeability of the pulmonary endothelium in rat lungs perfused for 60 min with buffered Hanks' bovine serum albumin medium. In one group of experiments, we examined the effect of H2O2 on the uptake and transport of cationized ferritin (CF) by endothelial cells in intra-acinar arteries, alveolar capillaries, and interlobular veins. The influence of the oxidant on endothelial adsorptive endocytic processes was assessed by measuring the density of ferritin particles in luminal vesicles, multivesicular bodies, and basal lamina. In a second group of experiments, we examined the effects of H2O2 on the fine structure and permeability to electron-dense macromolecules of arterial, microvascular, and venous endothelium. For this purpose, at the end of the 60-min perfusion with H2O2, CF was perfused to identify leaky vessels. We found that H2O2 caused a dose-dependent inhibition of transcytosis of CF in all vascular segments. At the lower dose of H2O2, inhibition of transcytotic activity was not associated with structural injury to the vascular endothelium or with elevation of wet-to-dry ratios. At the higher oxidant dose, inhibition of transcytosis was associated with leaky arterial endothelium and elevation of wet-to-dry ratios (6.44 +/- 0.12 vs. 5.64 +/- 0.16, P less than 0.02). The effects of H2)2 were prevented by adding catalase to the perfusate. The selective loss of structural integrity and leakiness of the arterial endothelium were diminished but not completely abolished by perfusing the oxidant retrograde from the venous side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alveolar epithelial barrier functions in ventilated perfused rabbit lungs

We employed ultrasonic nebulization for homogeneous alveolar tracer deposition into ventilated perfused rabbit lungs. (22)Na and (125)I-albumin transit kinetics were monitored on-line with gamma detectors placed around the lung and the perfusate reservoir. [(3)H]mannitol was measured by repetitive counting of perfusion fluid samples. Volume of the alveolar epithelial lining fluid was estimated with bronchoalveolar lavage with sodium-free isosmolar mannitol solutions. Sodium clearance rate was -2.2 +/- 0.3%/min. This rate was significantly reduced by preadministration of ouabain/amiloride and enhanced by pretreatment with aerosolized terbutaline. The (125)I-albumin clearance rate was -0.40 +/- 0.05%/min. The appearance of [(3)H]mannitol in the perfusate was not influenced by ouabain/amiloride or terbutaline but was markedly enhanced by pretreatment with aerosolized protamine. An epithelial lining fluid volume of 1.22 +/- 0.21 ml was calculated in control lungs. Fluid absorption rate was 1.23 microl x g lung weight(-1) x min(-1), which was blunted after pretreatment with ouabain/amiloride. We conclude that alveolar tracer loading by aerosolization is a feasible technique to assess alveolar epithelial barrier properties in aerated lungs. Data on active and passive sodium flux, paracellular solute transit, and net fluid absorption correspond well to those in previous studies in fluid-filled lungs; however, albumin clearance rates were markedly higher in the currently investigated aerated lungs.     

Ventilation-perfusion relationships in isolated blood-free perfused rabbit lungs.

The multiple inert gas elimination technique (MIGET) was applied to blood-free perfused isolated rabbit lungs. Commonly accepted criteria for reliability of the method were found to be fulfilled in this model. Ventilation-perfusion (VA/Q) distributions in isolated control lungs corresponded to those repeatedly detected under physiological conditions. In particular, a narrow unimodal dispersion of perfusate flow was observed: perfusion of low-VA/Q areas ranged below 1% and shunt flow approximately 2-3%; perfusion of high-VA/Q regions was not detected. Gas flow was characterized by narrow dispersion in the midrange-VA/Q areas. Application of a low level of PEEP (1 cmH2O) reduced shunt flow to less than 1%, and low-VA/Q areas were no longer noted. By using this PEEP-level, stable gas exchange conditions were maintained for greater than 5 h of extracorporeal perfusion. Graded embolization with small air bubbles caused a typical rightward shift (to higher VA/Q ratios) of mean ventilation, associated with the appearance of high-VA/Q regions and an increase in dead space ventilation. Mean perfusion was shifted leftward, and shunt flow was approximately doubled. Whole lung lavage with saline for washout of surfactant evoked a progressive manifold increase in shunt flow, accompanied by a moderate rise of perfusate flow to low-VA/Q areas. We conclude that the MIGET can be applied to isolated blood-free perfused rabbit lungs for assessment of gas exchange and that typical patterns of VA/Q mismatch are reproduced in this model.     

利用兔离体肺灌流模型评价肺微小血管通透性

目的准确、定量的评价肺微小血管通透性.方法利用兔离体肺灌流模型,采用肺重量分析法测定肺毛细血管滤过系数(Kf).结果肺毛细血管滤过系数测定值为4.78±0.73mg·min-1.cmH2O-1·g-1.结论这种利用离体肺灌流模型定量评价肺微小血管通透性的方法具有直接、测定准确的优点,对于了解肺的生理状态、评价急性肺损伤和肺水肿程度具有重要意义,是一种新型的实验方法.     

Endotoxin priming of thromboxane-related vasoconstrictor responses in perfused rabbit lungs

Steudel, Wolfgang, Hans-Joachim Krämer, DanielaDegner, Simone Rosseau, Hartwig Schütte, Dieter Walmrath, andWerner Seeger. Endotoxin priming of thromboxane-relatedvasoconstrictor responses in perfused rabbit lungs. J. Appl. Physiol. 83(1): 18-24, 1997.In priorstudies of perfused lungs, endotoxin priming markedly enhancedthromboxane (Tx) generation and Tx-mediated vasoconstriction inresponse to secondarily applied bacterial exotoxins. Thepresent study addressed this aspect in more detail by employingprecursor and intermediates of prostanoid synthesis and performingfunctional testing of vasoreactivity and measurement of productformation. Rabbit lungs were buffer perfused in theabsence or presence of 10 ng/ml endotoxin. Repetitive intravascularbolus applications of free arachidonic acid provoked constant pulmonaryarterial pressor responses and constant release reactions ofTxA₂ and prostaglandin (PG)I₂ in nonprimed lungs. Within60-90 min of endotoxin recirculation, which provoked progressiveliberation of tumor necrosis factor- but did not effect anyhemodynamic changes by itself, both pressor responses and prostanoidrelease markedly increased, and both events were fully blocked bycyclooxygenase (Cyclo) inhibition with acetylsalicylic acid(ASA). The unstable intermediatePGG₂ provoked moderate pressorresponses, again enhanced by preceding endotoxin priming and fullysuppressed by ASA. Vasoconstriction also occurred in response to thedirect Cyclo product PGH₂, again amplified after endotoxin pretreatment, together with markedly enhancedliberation of TxA₂ andPGI₂. In the presenceof ASA, the priming-related increase in pressor responses and theprostanoid formation were blocked, but baseline vasoconstrictorresponses corresponding to those in nonprimed lungs were maintained.Pressor responses to the stable Tx analog U-46619 were notsignificantly increased by endotoxin pretreatment, but some generationof TxA₂ andPGI₂ was also noted under theseconditions. We conclude that endotoxin priming exerts profound effectson the lung vascular prostanoid metabolism, increasing the readiness toreact with Tx-mediated vasoconstrictor responses to various stimuli,suggesting that enhanced Cyclo activity is an important underlyingevent.

     

Removal of atrial natriuretic peptide by perfused rabbit lungs in situ

Removal of iodinated 28-amino acid atrial natriuretic peptide ([125I]ANP) by rabbit lungs was measured by indicator-dilution methods. After bolus injection of 6.5 pmoles of [125I]ANP, 66.9 +/- 2.9% was removed in a single pass through the lungs. Removal was unaltered by a kininase II inhibitor but was reversibly decreased by unlabelled ANP. Thus the lungs can remove ANP from the pulmonary circulation by a mechanism that does not involve hydrolysis by kininase II. Lungs therefore may be involved in regulating systemic concentrations and hence renal and other actions of ANP.     

Flow-mediated release of nitric oxide in isolated, perfused rabbit lungs.

The effects of changing perfusate flow on lung nitric oxide (NO) production and pulmonary arterial pressure (Ppa) were tested during normoxia and hypoxia and after N(G)-monomethyl-L-arginine (L-NMMA) treatment during normoxia in both blood- and buffer-perfused rabbit lungs. Exhaled NO (eNO) was unaltered by changing perfusate flow in blood-perfused lungs. In buffer-perfused lungs, bolus injections of ACh into the pulmonary artery evoked a transient increase in eNO from 67 +/- 3 (SE) to 83 +/- 7 parts/billion with decrease in Ppa, whereas perfusate NO metabolites (pNOx) remained unchanged. Stepwise increments in flow from 25 to 150 ml/min caused corresponding stepwise elevations in eNO production (46 +/- 2 to 73 +/- 3 nl/min) without changes in pNOx during normoxia. Despite a reduction in the baseline level of eNO, flow-dependent increases in eNO were still observed during hypoxia. L-NMMA caused declines in both eNO and pNOx with a rise in Ppa. Pulmonary vascular conductance progressively increased with increasing flow during normoxia and hypoxia. However, L-NMMA blocked the flow-dependent increase in conductance over the range of 50-150 ml/min of flow. In the more physiological conditions of blood perfusion, eNO does not reflect endothelial NO production. However, from the buffer perfusion study, we suggest that endothelial NO production secondary to increasing flow, may contribute to capillary recruitment and/or shear stress-induced vasodilation.     

Anatomical description of the renal arteries and veins in the European rabbit

The aim of this study was to describe origin, localisation and variations of renal arteries and veins in the rabbit. The study was carried out on 40 adult European rabbits. We prepared corrosion casts of the rabbit arterial and venous system. Spofacryl was used as the casting medium. In 75% of cases the origin of arteriae renales was located at the level of the third lumbar vertebra and in remaining 25% of cases arteria renalis dextra branched off at the level of the second lumbar vertebra. In 10% of cases we observed that the number of arteria renalis sinistra was doubled. We recorded also in one case the presence of arteria renalis accessoria for ren dexter. In 10% of cases we observed that the number of vena renalis sinistra was doubled. In 5% of cases two venae renales sinistrae arose from the kidney and subsequently, about 1 cm from opening to vena cava caudalis, they united to form a single vein. In 5% of cases two venae renales sinistrae arose from the kidney and subsequently, approximately 1 cm away from hilus renalis, they united. The obtained variations of the number of renal arteries were partially homologous to the human, but variations of renal veins were localized on the other side as in human.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. ¹⁴C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0–4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE₂ and its 15-keto-derivates, but also PGF_2α and its 15-keto-derivates, TXB₂ and 6-keto-PGF_1α were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE₂ from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. 14C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0-4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE2 and its 15-keto-derivates, but also PGF2 alpha and its 15-keto-derivates, TXB2 and 6-keto-PGF1 alpha were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE2 from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

Comparative evaluation of sequestration of polyamines by perfused rabbit and rat lungs

Differences were observed in the sequestration of polyamines putrescine, spermidine and spermine by isolated, ventilated, perfused rat and rabbit lungs, former being able to accumulate more polyamines compared to the latter. Steady state equilibrium was reached earlier for spermine in rat. Isolated ventilated lungs were perfused with harmaline and ouabain, inhibitors known to inhibit the sodium pump at a maximum concentration of 1 mM for rabbit lungs and 0.4 and 0.2 mM for rat lungs, respectively. They did not affect the uptake of polyamines by rat lung but decreased the uptake of putrescine by rabbit lung. Decreased sodium (50 meq/L) in the perfusate increased the uptake of spermine and spermidine by rabbit lung but again showed no effect with rat lung. However, the uptake of polyamines by isolated ventilated rat and rabbit lungs perfused for 60 min with these compounds was linear over the entire range of high concentrations studied. These results suggest that the major uptake process of polyamines by intact lungs of both animal species is primarily by simple diffusion. HPLC analysis of the perfusate and lungs from both animal species post-perfusion indicated no detectable metabolites of the polyamines.     

Apoptosis in microvascular endothelial cells of perfused rabbit lungs with acute hydrostatic edema.

We test the hypothesis that microvascular endothelial cells may undergo apoptosis in response to acute pulmonary venous hypertension. The isolated rabbit lungs were perfused in situ for 4 h with left atrial pressure of 0, 10, or 20 mmHg at a constant blood flow. Edema formation was monitored by lung weight gain. To assay for apoptosis, we performed agarose gel electrophoresis of DNA, in situ nick end labeling of DNA strand breaks, and electron microscopy. We also examined the levels of expression of Bcl-2, a suppressor of apoptosis, in microvascular endothelial cells using an immunohistochemical technique. In a vascular pressure-dependent fashion, we found apoptosis in endothelial cells of alveolar septal capillaries, as well as expression of Bcl-2 in arteriolar and venular endothelial cells. We conclude that acute pulmonary venous hypertension induces apoptosis in capillary endothelial cells but not in arteriolar and venular endothelial cells, suggesting that microvascular endothelial cell apoptosis is dependent on the levels of Bcl-2 expression and influences the formation or resolution of acute hydrostatic lung edema.